Bacillus subtilis, an endospore-forming bacterium, is, after Escherichia coli, nature's best studied organism and has proved to be a useful model for other gram-positive bacteria, pathogens and non-pathogens alike. Endosporulation in Bacillus and Clostridium spp. is a response to severe nutrient limitation. Thus, the metabolic signals that the cell perceives are a crucial component of the regulation of this differentiation process. In recent prior work, the B. subtilis CodY protein has been identified as a metabolite-sensing repressor that controls a number of genes that are turned on when cells experience nutrient deprivation. Some of these genes must be critical for sporulation, because a codY mutant sporulates under conditions of nutrient excess. It has long been recognized that the intracellular concentration of GTP drops when cells make the transition from rapid exponential growth to stationary phase. CodY has now been shown to respond to the intracellular level of GTP, losing activity as a repressor as the GTP concentration drops. CodY also responds to isoleucine and valine, two of the branched chain amino acids. These novel findings suggest that CodY plays a surprisingly general role in cellular metabolism and differentiation. In fact, microarray analysis has shown that hundreds of genes respond to the absence of CodY and are thus either direct or indirect targets of the regulatory protein. This proposal seeks to address several fundamental questions about the role of CodY and the mechanism by which it regulates transcription. The specific aims of the proposal are: (i) to determine the role of CodY in initiation of sporulation; (ii) to determine the global role of CodY in metabolism; (iii) to determine the molecular architecture of CodY and its interaction with effectors; (iv) to characterize the interaction between CodY and its DNA targets; and, (v) to identify a hypothetical second signaling pathway that mediates the switch from stationary phase to sporulation. The results of this research have the potential to resolve longstanding mysteries, such as why the onset of sporulation is accompanied by a transient drop in the GTP pool, and to provide new insight into the metabolic regulation of stationary phase and early sporulation events. Given the existence in other gram-positive bacteria of proteins that are remarkably similar to CodY, it is likely that the findings of this project will have general application to a broad group of important prokaryotes.